Botulinum neurotoxins are a family of seven (serotypes A-G) pharmacologically similar but serologically distinct potent neuroparalytic proteins produced by strains of Clostridium botulinum. They are recognized as the most poisonous toxins known and are designated by CDC as Category A biothreat agents. Vaccines provide the only comprehensive means of protection against botulinum intoxication. However, there are currently no FDA-approved botulinum vaccines, and the existing pentavalent botulinum toxoid (PBT) vaccine (an IND product for the last 35 years) was last formulated in 1990s from components manufactured in the early 1970s. The supply of PBT is extremely limited, and the product appears to be decreasing in potency. There are other recombinant botulinum vaccines under development, and they are mostly based on the heavy chain (Hc) fragment of the toxin molecule. Intrinsic properties of the Hc fragment present challenges to the use of Hc as a vaccine candidate: issues associated with vaccine formulation, stability and protection against engineered toxins. To address the urgent unmet medical need for an effective botulinum vaccine, Emergent Product Development Gaithersburg (Emergent) has internally funded the development of a recombinant botulinum vaccine (rBot) based on the LHN fragment of the toxin molecule. The LHN-based vaccine candidate offers advantages over other recombinant approaches, including improved stability, cross- protection against toxin subtypes and ease of formulation into a multivalent vaccine. The overall objective of the proposed work is to develop a safe and efficacious vaccine against botulinum neurotoxin serotypes A and B, which are viewed as the most prominent biothreat serotypes. Additional serotypes can be added to the vaccine formulation later if the U.S. Government so desires. Immunogenicity and efficacy testing in guinea pigs and mice has shown that this approach produces a strong and protective immune response and is effective against toxin subtypes. To undertake this research program, Emergent has partnered with the Health Protection Agency (Porton Down, UK). Together, these organizations offer a wealth of experience in botulinum vaccine development. Advanced process development has been completed, and a consistent and commercially viable manufacturing process has been developed that produces highly purified and soluble LHN proteins. The specific aims of this grant include activities required for entrance into Phase 1 clinical trial: 1. Pre-clinical Testing of Monovalent LHN/A&B and Bivalent LHN/AB Vaccine 2. Manufacture of cGMP Clinical Lots (Monovalent LHN/A, B and Bivalent LHN/AB Final Product) 3. Stability Testing of cGMP Clinical Lots (LHN/A, B and LHN/AB)